Protein identification by peptide mass fingerprinting (PMF) using the MS/MS techniques (tandem mass spectrometry) post-source decay (PSD) or collision-induced dissociation (CID) is based on the comparison of experimentally derived data with theoretically calculated masses in databases (Gevaert et al. Electrophoresis 2001). However, since the genome sequence of most organisms is still incomplete, information on particular proteins is not included in existing databases. In addition, even if relevant protein information existed in the databases, different modifications such as post-translational modifications can hamper identification of a portion or a complete amino acid sequence. Therefore, complete determination of primary protein structure requires detection of an amino acid sequence with the minimal use of databases, i.e., de novo peptide and protein sequencing (H. Steen et al. Mol. Cell. Biol. 2004). The latter is based on tandem mass spectrometry, MS/MS or PSD experiments. In order to facilitate interpretation of complex spectra, peptides are chemically derivatized by appropriate reagents that either almost exclusively or in most of the cases give rise to one series of fragment ions. Mass difference between consecutive signals reveals amino acid sequence. Keough's idea of binding acidic group with N-terminus of peptide has proved successful. Such derivatized peptide bearing positive and negative charge at the same time can be depicted by the following formula: −O3S—C6H5-A1-A2-A3-A4-A5-X+, where A stands for any amino acid, and X for lysine or arginine (T. Keough et al. Proc. Natl. Acad. Sci. 1999, T. Keough et al. Rapid Commun. Mass Spectrom. 2000).
If derivatization by acidic group at N-terminus is performed on lysine peptide, then ε-amino group of lysine is chemically modified with imidazole group (so called Lys-tag) as to increase the portion of these ions in gas phase and at the same time to enable selective reaction of acidic group with N-terminus (R. L. Beardsley et al. Anal. Chem. 2002). This procedure is known as guanidination. Since the most basic peptide group is already protonated (arginine or guanidinated lysine), an additional proton needed for molecule ionization randomly selects the site of protonation. Peptide bond breaks at the site of protonation resulting in the formation of b- and y-ion series products. However, fragments comprising SO3− will not be detected during the MS/MS scan of positive ions due to their instability, so that mass spectrum reveals only y-ion series signals without mass increments, as if derivatization of N-terminus has not been performed (P. Conrotto et al. J. Biomol. Techn. 2005, P. Conrotto et al. Am. Biotechnol. Lab. 2006).
Current procedures of peptide or protein derivatization yield better results in comparison with complex analysis of non-derivatized peptides. However, obtained spectra have not demonstrated sufficiently intense ion signals necessary for detection of amino acids in the spectrum. Furthermore, high level of background noise additionally contributed to imprecise amino acid detection in the spectrum and consequently to imprecise detection of amino acid sequence, i.e., to imprecise peptide or protein identification.
The most common sulfonyl group-containing reagents used in peptide or protein derivatization are 2-sulfobenzoic acid (T. Keough et al. Proc. Natl. Acad. Sci. 1999), sulfo-NHS esters (N-hydroxysuccinimide, NHS; W. R. Allery et al. J. Prot. Research 2007), and 4-sulfophenyl isothiocyanate (P. Chen et al. Rapid Commun. Mass Spectrom. 2004), which relatively quickly (up to 30 minutes) modify peptide and make it amenable to sequencing. Current literature describes sulpho-derivatization reagents based on the following reactive groups: isothiocyano (Y. H. Lee et al. Proteomics 2004), isocyano (P. Conrotto et al. J. Biomol. Techn. 2005), cyclic anhydride (T. Keough et al. Proc. Natl. Acad. Sci. 1999), and N-Hydroxysuccinimide (W. R. Allery et al. J. Prot Research 2007).
In the state of the art there are several patents/patent applications that reveal different methods of detection of amino acid sequence and/or identification of proteins, peptides. However, none of the below stated documents reveal derivatization of peptides or proteins in such a way as to produce negative ions that might be detected in negative MS/MS spectrum.
WO2000043792 describes a procedure based on the use of the compounds with one or more acidic groups with pKa lower than 2 for derivatization of peptide N-terminus. Furthermore, this invention implies that derivatized y-ions are used for analysis of fragments by mass spectrometry, which are devoid of a- and b-ions. Although derivatives of disulfonic acids are also mentioned as acidic groups, derivatization procedure described in this document is not used for spectra analysis in negative MS/MS mode.
WO2002008767 refers to derivatization of lysine-containing peptides. As in the document mentioned above, derivatization procedure in this document is not used for spectra analysis in negative MS/MS mode. Furthermore, this document refers to guanidination, i.e., imidolization of lysine, which is not necessary with the use of the subject invention.
WO2002095412 describes the use of water-stable reagents for peptide derivatization. The reagents comprise one or more sulfonyl groups bound with activated acidic group via aliphatic or aromatic linkage. Activated acid derivatives described in this patent application are acid esters, anhydrides of organic and inorganic acids. The activated acidic moiety particularly mentioned in this patent is NHS ester, allowing for all procedure steps to be carried out under aqueous conditions. The four basic steps in the subject invention include guanidination of lysine. Furthermore, this invention refers to the y-ions analysis in positive mode of operation of mass spectrometer. Derivatization procedure in this document is not used for spectra analysis in negative MS/MS mode.
WO2002095419 holds priority of the application WO2002095412, and, therefore, shares a high level of similarity with the latter patent. In comparison with aforementioned patent, it has been added that polypeptides are immobilized on the solid support, at least in the step “a”. This means that complete derivatization procedure is not carried out in solution, the latter being the case with subject inventions. In addition, derivatization procedure in the aforementioned document is not used to analyse spectra in negative MS/MS mode.
European patent application EP 1561755 describes compounds with disulfide bond that react with peptide N-terminus followed by disulfide bond cleavage under oxidation or reduction conditions resulting in the formation of sulfonic acid derivatives. Functional group of disulfide compound that reacts with N-terminus was chosen among carboxyl group, isothiocyanate, succinimidyl oxycarbonyl groups, p-nitrophenoloxy carbonyl groups, pentafluorophenyloxy carbonyl groups, and tetrafluorosulpho phenyloxycarbonyl groups. Subject invention is used for detection of amino acid sequence by y-ions analysis in positive mode of operation of mass spectrometer. In this invention, guanidination of lysine is also necessary. In addition, derivatization procedure in the aforementioned document is not used for spectra analysis in negative MS/MS mode.
WO2005078451 improves previous methods in such a way that removal of unmodified portions of peptides from the solution by ion exchange precedes the analysis of fragments in positive mode of operation of mass spectrometer. This procedure is used after any chemically-aided peptide derivatization.
Accordingly, the conventional procedure of peptide derivatization in the state of the art was carried out by introduction of sulfonyl groups to the N-terminus. N-terminus derivatized in this manner becomes negatively charged. Positively charged C-terminus is a counterbalance to negatively charged N-terminus resulting in the formation of the so called zwitterion, i.e., charge of derivatized protein or peptide equals zero. In further ionization procedure in mass spectrometer used in the state of the art, proton was added to peptide/protein reducing the energy necessary for peptide bonds cleavage, which produced mostly b- and y-ions. Since b-ions would be neutral due to negative charge at N-terminus, only positive y-ions in positive mode of operation of mass spectrometer were analysed in the state of the art.